Modulation of Cannabinoid Receptor Activity by Allosteric Modulators, Inverse Agonists and Receptor Binding Partners

The regulation of G protein coupled receptors (GPCRs) plays a fundamental role in physiologic homeostasis. There are two cannabinoid receptors: CB1, found on neurons of the central and peripheral nervous system and regulate neuromodulatory processes and CB2, found in peripheral tissues, particularly in immune tissues. In this research, three different modulation mechanisms of the cannabinoid receptors are studied. Chapter 2 focuses on the structure-activity relationships of novel allosteric modulators from the indole-2-carboxamide class of compounds. These novel allosteric modulators were optimized for their KB (binding affinity to the allosteric site) and their cooperativity factor ? (magnitude by which affinity of orthosteric ligand is changed). Although these positive allosteric modulators enhanced orthosteric CP55, 950 agonist affinity and decreased SR141716A inverse agonist affinity to CB1 consistent with an active receptor conformation, they antagonized basal and agonist-induced G protein coupling. Chapter 3 focuses on the characterization of a group of new inverse agonists from the class of benzhydryl piperazine analogs. These compounds exhibit high nanomolar binding affinities to CB1, antagonize basal as well as agonist-induced G protein coupling and increase receptor cell surface localization, consistent with inverse agonist behavior. Docking and mutational analyses revealed SR141716A-like interactions in the CB1 binding pocket. However, this benzhydryl piperazine scaffold is structurally distinct from first generation CB1 inverse agonists and holds promise for developing peripherally active CB1 inverse agonists with fewer psychiatric side effects. Chapter 4 focuses on the development and optimization of a novel technique using the photo-cross-linking unnatural amino acid p-benzoyl-L-phenylalanine (pBpa) in live cells. This will elucidate the binding partners of the CB2 receptor at defined locations and time points in real-time using mass spectrometry. To this end, I determined the best detergent to solubilize CB2, the ratios of DNA to tRNA and pBpa-synthetase needed, the optimal concentration of pBpa in the media and the ideal UV exposure times needed for the efficient incorporation of pBpa into the full-length CB2 receptor. Additionally, pilot experiments using different CB2 ligands and varying treatment times were performed and possible CB2-binding partner complexes were observed. These results will aid in the discovery of more effective and selective GPCR ligands.